Flow cell array and the utilization thereof for multianalyte determination

ABSTRACT

A one- or two-dimensional arrangement of flow cells is provided, as part of an array of sample compartments, with at least one inlet and outlet for each sample compartment, formed by a base plate and a body, with an arrangement of spatial recesses corresponding to the (geometrical) arrangement of the sample compartments, combined with the base plate. The arrangement allows for supplying to or removing from the sample compartments, which can be arranged at a high quantity on a small base area, even very small amounts of samples or reagents. An arrangement of one or more sample compartments includes a base plate and a body combined with the base plate in such a way that one or more recesses for generation of one or more flow cells fluidically sealed against one another, each with at least one inlet and one outlet, are formed between the base plate and the body, wherein at least one outlet of each flow cell is joined with a reservoir fluidically connected with the flow cell, the reservoir being operable to receive liquid exiting the flow cell, besides methods for its manufacturing and their use.

The invention is related to a one- or two-dimensional arrangement offlow cells, as part of an array of sample compartments, with at leastone inlet and outlet for each sample compartment, formed by a base plateand a body, with an arrangement of spatial recesses corresponding to the(geometrical) arrangement of the sample compartments, combined with thebase plate. The arrangement allows for supplying to or removing from thesample compartments, which can be arranged at a high quantity on a smallbase area, even very small amounts of samples or reagents. For eachsample compartment at least one reservoir for receiving liquids to beremoved from the respective sample compartment is integrated in thearrangement, the peripheral manifold of liquid supply and removal meanscan be simplified significantly in comparison to conventional technicalsolutions.

For the determination of a single analyte in a sample, especially fordiagnostics, numerous arrangements have become known, wherein thetransport of samples or reagents is performed using capillary forces.Thus the use of pumps for fluidic transport can often be avoided. InU.S. Pat. No. 5,019,351 a special embodiment of such a device isdescribed, wherein a reaction capillary with an inlet end and an outletend is arranged between a base plate and a cover plate, the inlet endbeing fluidically connected with a region dedicated for receiving asample and mixing the sample with reagents, and wherein the reactioncapillary is divided into an upstream region and a downstream region,with increasing thickness of the reaction capillary from the upstreamregion towards the downstream region. With this arrangement constant,reproducible flow conditions shall be achieved within the reactioncapillary. Additionally, embodiments are described in that patentdisclosure, wherein the outlet end of the reaction capillary isconnected with a region for collecting exiting liquid. Thereby, allcomponents of the structure, except for the inlet region, are providedin a continuous channel or capillary system, respectively, between thecommon base plate and a common cover plate. The essential technicalbackground of the patent disclosure is based on liquid transport by thecapillary effect.

However, especially because of the necessary large space requirements,such an arrangement pf capillary-type flow cells is hardly suited for anapplication in combination with a multitude of measurement areas, forthe determination of different analytes or the analysis of differentsamples, on a common support. Accordingly, there are not any hintstowards array-type arrangements of several flow cells of this type inU.S. Pat. No. 5,019,351. Additionally, a removal of liquid havingentered the collection region is not possible.

For the determination of a multitude of analytes, currently mainly suchmethods are used, wherein the determination of different analytes isperformed in discrete sample compartments or “wells” of so-calledmicrotiter plates. The most common are plates with a pitch (geometricalarrangements in rows and columns) of 8×12 wells on a footprint oftypically about 8 cm×12 cm (see, for example, Corning Costar catalogueNo. 3370, 1999), wherein a volume of some hundred microliters isrequired for filling a single well. It would be desirable for manyapplications, however, to reduce the sample volume considerably, notonly for a reduction of necessary amounts of samples and reagents, whichmight be available only at small amounts, but especially for a reductionof the diffusion pathways and thus of the assay times, for assayswherein biological or biochemical or synthetic recognition elements forthe recognition of one or more analytes in a sample are immobilized onthe inner wall of a sample compartment.

In case of open sample compartments arranged in an array, derived fromthe classical microtiter plate, a well-known technical solution for areduction of the volumes of the individual sample compartments wasprovided by arranging a larger number of wells on the same base area(foot print), for example 384 (see, for example, Corning Costarcatalogue No. 3702, 1999) or 1536 instead of classically 96 wells. Thisapproach has the advantage that the instruments and laboratory robots,established as the industrial standard, can be further used at least fora part of the necessary steps of sample manipulation, such as samplesupply and removal. Other known technical solutions were provided byabandoning the classical plate foot print and adapting the size of theindividual wells to the sample volumes required for a certainapplication, exclusively. Such arrangements became known under the name“nanotiter plates”, the volumes of the individual sample compartmentspartially being only some nanoliters. However, this technical solutionrequires the abandonment of the currently wide-spread laboratory robotsdesigned for the classical microtiter plate standard.

Along with the reduction of the size of the individual, open samplecompartments and/or with the reduction of the thickness of the liquidlayer on the base surface, desired for a reduction of the diffusionpathways, however, the increasing effect of liquid evaporation duringthe time of an assay becomes a more and more severe problem that has tobe taken care of.

Said problem of evaporation can be avoided by using sample compartmentsthat are closed, except for openings for sample inlet and outlet.

Such through-flow cells, operable for receiving a liquid sample and orreagents in a single flush or continuously, are known for the case of asingle measurement area in contact with a sample compartment.

In U.S. Pat. No. 5,747,274, measurement arrangements and methods for theearly recognition of a cardiac infarction, upon determination of severalfrom at least three infarction markers, are described, wherein thedetermination of these markers can be performed in individual samplecompartments or in a common sample compartment, a single (common) samplecompartment being provided, according to the disclosure for the lattercase, as a continuous flow channel, one demarcation of which beingformed, for example, by a membrane, whereon antibodies for the threedifferent markers are immobilized. However, there are no hints for anarrangement of several sample compartments or flow channels of this typeon a common support. Additionally, there is no geometrical informationconcerning the size of the measurement areas.

Optical methods, for example based on the determination of the change ofan absorbance or of a luminescence, have been developed for analytedetermination in one or more samples to an increasing extent in thepast, as such methods can be performed without contact and withoutsignificant effect on the sample itself. The classical measurementmethods, such as absorption or fluorescence measurements, are generallybased on the direct illumination of a sample volume in a samplecompartment or of a measurement area on the inner wall of thecompartment of a liquid sample. As a disadvantage of theseconfigurations, in general a significant part of the surroundings,besides the excitation volume or excitation area where a signal foranalyte determination shall be generated, is illuminated by excitationlight, which can lead to disadvantageous generation of disturbingbackground signals.

For achieving lower detection limits, numerous measurement arrangementshave been developed, wherein the determination of an analyte is based onits interaction with the evanescent field, which is associated withlight guiding in an optical waveguide, wherein biochemical or biologicalrecognition elements for the specific recognition and binding of theanalyte molecules are immobilized on the surface of the waveguide. Whena light wave is coupled into an optical waveguide surrounded byoptically rarer media, i.e. media of lower refractive index, the lightwave is guided by total reflection at the interfaces of the waveguidinglayer. In that arrangement, a fraction of the electromagnetic energypenetrates the media of lower refractive index. This portion is termedthe evanescent (=decaying) field. The strength of the evanescent fielddepends to a very great extent on the thickness of the waveguiding layeritself and on the ratio of the refractive indices of the waveguidinglayer and of the media surrounding it. In the case of thin waveguides,i.e. layer thicknesses that are the same as or smaller than thewavelength of the light to be guided, discrete modes of the guided lightcan be distinguished. As an advantage of such methods, the interactionwith the analyte is limited to the penetration depth of the evanescentfield into the adjacent medium, being of the order of some hundrednanometers, and interfering signals from the depth of the (bulk) mediumcan be mainly avoided. The first proposed measurement arrangements ofthis type were based on highly multi-modal, self-supporting single-layerwaveguides, such as fibers or plates of transparent plastics or glass,with thicknesses from some hundred micrometers up to severalmillimeters.

In U.S. Pat. No. 4,978,503, a measurement arrangement is disclosed,wherein a liquid sample is drawn into a cavity by capillary forces,wherein an optically transparent side wall is provided as aself-supporting multimode waveguide, wherein at least on a part of thesurface of that side wall (“patch”), facing the cavity, a biochemicalrecognition element for the recognition and binding of an analyte from asample is immobilized. As a disadvantage of this arrangement, anexchange of the liquid drawn into the capillary is not provided for,such an exchange being for example desirable for a multi-step assay.

In WO 94/27137, measurement arrangements are disclosed, wherein“patches” with different recognition elements, for the determination ofdifferent analytes, are immobilized on a self-supporting opticalsubstrate waveguide (single-layer waveguide), excitation light beingincoupled at the distal surfaces (“front face” or “distal end”coupling), wherein laterally selective immobilization is performed usingphoto-activatable cross-linkers. According to the disclosure, severalpatches can be arranged row-wise in common, parallel flow channels orsample compartments, wherein the parallel flow channels or samplecompartments extend over the whole length of the range on the waveguideused as a sensor, in order to avoid an impairment of light guiding inthe waveguide. However, there are no hints to a two-dimensionalintegration of multiple patches and sample compartments. In a similararrangement disclosed in WO 97/35203, several embodiments of anarrangement are described, wherein different recognition elements forthe determination of different analytes are immobilized in separate,parallel flow channels or sample compartments for the sample and forcalibration solutions of low and, optionally in addition, of highanalyte concentration. Again, no hint to two-dimensional arrangements isgiven.

For an improvement of the sensitivity and simultaneously for an easiermanufacturing in mass production, planar thin-film waveguides have beenproposed. In the simplest case, a planar thin-film waveguide consists ofa three-layer system: support material (substrate), waveguiding layer,superstrate (respectively the sample to be analyzed), wherein thewaveguiding layer has the highest refractive index. Additionalintermediate layers can further improve the action of the planarwaveguide.

Several methods for the incoupling of excitation light into a planarwaveguide are known. The methods used earliest were based on front facecoupling or prism coupling, wherein generally a liquid is introducedbetween the prism and the waveguide, in order to reduce reflections dueto air gaps. These two methods are mainly suited with respect towaveguides of relatively large layer thickness, i.e. especiallyself-supporting waveguides, and with respect to waveguides with arefractive index significantly below 2. For incoupling of excitationlight into very thin waveguiding layers of high refractive index,however, the use of coupling gratings is a significantly more elegantmethod.

Different methods of analyte determination in the evanescent field oflightwaves guided in optical film waveguides can be distinguished. Basedon the applied measurement principle, for example, it can bedistinguished between fluorescence, or more general luminescencemethods, on one side and refractive methods on the other side. In thiscontext methods for generation of surface plasmon resonance in a thinmetal layer on a dielectric layer of lower refractive index can beincluded in the group of refractive methods, if the resonance angle ofthe launched excitation light for generation of the surface plasmonresonance is taken as the quantity to be measured. Surface plasmonresonance can also be used for the amplification of a luminescence orthe improvement of the signal-to-background ratios in a luminescencemeasurement. The conditions for generation of a surface plasmonresonance and the combination with luminescence measurements, as well aswith waveguiding structures, are described in the literature, forexample in U.S. Pat. No. 5,478,755, U.S. Pat. No. 5,841,143, U.S. Pat.No. 5,006,716, and U.S. Pat. No. 4,649,280.

In this application, the term “luminescence” means the spontaneousemission of photons in the range from ultraviolet to infrared, afteroptical or other than optical excitation, such as electrical or chemicalor biochemical or thermal excitation. For example, chemiluminescence,bioluminescence, electroluminescence, and especially fluorescence andphosphorescence are included under the term “luminescence”.

In case of the refractive measurement methods, the change of theeffective refractive index resulting from molecular adsorption to ordesorption from the waveguide is used for analyte detection. This changeof the effective refractive index is determined, in case of gratingcoupler sensors, from changes of the coupling angle for the in- orout-coupling of light into or out of the grating coupler sensor, in caseof interferometric sensors from changes of the phase difference betweenmeasurement light guided in a sensing branch and a referencing branch ofthe interferometer.

The aforesaid refractive methods have the advantage that they can beapplied without using additional marker molecules, so-called molecularlabels. The disadvantage of these label-free methods, however, is thatthe achievable detection limits are limited to pico- to nanomolarconcentration ranges, dependent on the molecular weight of the analyte,due to lower selectivity of the measurement principle, which is notsufficient for many applications of modern trace analysis, for examplefor diagnostic applications. For achieving lower detection limits,luminescence-based methods appear as more adequate, because of higherselectivity of signal generation. In this arrangement, luminescenceexcitation is limited to the penetration depth of the evanescent fieldinto the medium of lower refractive index, i.e to the immediateproximity of the waveguiding area, with a penetration depth of the orderof some hundred nanometers into the medium. This principle is calledevanescent luminescence excitation.

By means of highly refractive thin-film waveguides, based on an onlysome hundred nanometers thin waveguiding film on a transparent supportmaterial, the sensitivity could be increased considerably during thelast years. In WO 95/33197, for example, a method is described, whereinthe excitation light is coupled into the waveguiding film by a reliefgrating as a diffractive optical element. The isotropically emittedluminescence from substances capable of luminescence, that are locatedwithin the penetration depth of the evanescent field, is measured byadequate measurement devices, such as photodiodes, photomultipliers orCCD cameras. The portion of evanescently excited radiation, that hasback-coupled into the waveguide, can also be out-coupled by adiffractive optical element, like a grating, and be measured. Thismethod is described, for example, in WO 95/33198.

For performing, simultaneously or sequentially, exclusivelyluminescence-based, multiple measurements with essentially monomodal,planar anorganic waveguides, for example in the specification WO96/35940, arrangements (arrays) have been proposed, wherein at least twodiscrete waveguiding areas are provided on one sensor platform, suchthat the excitation light guided in one waveguiding area is separatedfrom other waveguiding areas.

In the spirit of this invention, spatially separated measurement areas(d) (according to FIG. 4) shall be defined by the area that is occupiedby biological or biochemical or synthetic recognition elementsimmobilized thereon, for recognition of one or multiple analytes in aliquid sample. These areas can have any geometry, for example the formof dots, circles, rectangles, triangles, ellipses or lines.

In WO 98/22799, arrangements of sample compartments for measurementconfigurations for the determination of luminescence excited in theevanescent field of a planar waveguide are proposed, wherein thegratings used for incoupling of the excitation light are covered by thematerial forming the side walls of the sample compartments. Thus, anychange of the incoupling conditions can be avoided. This is, however,associated with very high requirements on the transparency, freedom offluorescence and a refractive index of the side wall material as low aspossible, which combination of requirements can hardly be satisfiedsimultaneously.

As a disadvantage, all devices described in the known technical state ofthe art do either not allow for an exchange of sample or reagent liquidsor require relatively complex arrangements for such an exchange, namelythe interfacing to fluidically sealing supplies and tube connections,for the supply and removal of liquid samples and reagents, especiallyimpeding an automated operation and exchange of combinations of baseplates, used as sensor platforms, with bodies placed thereon, forproviding flow cells, for single use operation, or requiring complextechnical solutions.

Therefore, there is a need for a simple arrangement of flow cells,especially in the form of one- or two-dimensional arrays, wherein thesupply of sample solutions and reagents and the removal of exitingsample and reagent fluids can be simplified significantly, withoutimpairing the measurement accuracy in a negative way.

Surprisingly, it now has been found, that a multitude of samplecompartments can be provided on a small base area, without therequirement of a complex system of peripheral supply and removal linesfor an automated supply and removal of samples and reagents, thanks tothe design of sample compartments according to the invention, the samplecompartments being each provided with a reservoir, for example in formof a recess in an exterior sample compartment wall, for receivingliquids exiting the sample compartments. Simultaneously, the volumecapacities of the individual sample compartments can be kept low, evenwhen relatively large base areas of the sample compartments on a baseplate are required for generation of a sufficiently high measurementsignal, because the sample compartments are closed except for inlet andoutlet openings for sample and reagent supply and removal, and becausethe height of a sample compartment, i.e. the distance between a baseplate and the opposite demarcation of a recess in a body combined withthe base plate, can be kept very small, i.e. even below 100 μm.

Due to this arrangement, even very low sample volumes can beadministered and kept constant. Due to the possible small volumes, theflow cells provided by the arrangement according to the invention can becleaned very efficiently by displacement washing. If biological orbiochemical or synthetic recognition elements for the determination ofan analyte are immobilized on the base plate, as described below forsome embodiments of an arrangement according to the invention, theend-point of diffusion-controlled analyte binding is reached fast, dueto only short diffusion pathways towards the analyzing surface, which isanother advantage. It is also favorable, that a signal caused by theanalyte binding is essentially independent of the total volume of asupplied sample, as long as at least the flow cell, as part of anarrangement according to the invention, is filled completely. The flowcells being closed except for inlets and outlets, an evaporation ofliquid can mainly be avoided, allowing for operation of the arrangementalso at temperatures significantly above room temperature. In summary,the present invention thus provides significant advantages in comparisonto the known state of the art.

The subject of the invention is an arrangement of one or more samplecompartments in a one- or two-dimensional array, comprising a base plateand a body combined with the base plate in such a way, that an array ofrecesses for generation of an array of flow cells fluidically sealedagainst one another, each with at least one inlet and one outlet, areformed between the base plate and the body, wherein at least one outletof each flow cell is joined with a reservoir fluidically connected withthe flow cell, the reservoir being operable to receive liquid exitingthe flow cell.

Thereby, the reservoir for receiving liquid exiting the flow cell isformed as a recess in the exterior wall of the body combined with thebase plate.

The arrangement according to the invention allows for supplyingsimultaneously, locally addressed, numerous different sample and reagentsolutions to different sample compartments, without the necessity ofremoving liquids that may have been supplied in advance. This does notrequire fixed tubing connections, but can be performed, for exampleusing the squirt of a dispenser, which can be directed to the inlet of aflow cell of the arrangement according to the invention. The reservoirsfor receiving liquids exiting from the flow cells being integrated inthe arrangement, exit tubes and their connections, which would berequired otherwise, are not needed. Thus, a multitude of flow cells foranalysis of different samples can be integrated on a minimum base area.

For the simultaneous supply of samples or reagents to a multitude ofsample compartments, multi-channel pipettors for manual or automatedreagent administration can be used, wherein the individual pipettes arearranged in one- or two-dimensional arrays, provided that the inlets ofthe arrangement of sample compartments according to the invention arearranged in the same pitch (geometrical arrangement in rows and/orcolumns). Preferably, therefore, the pitch of the arrangementcorresponds to the pitch of the wells of a standard microtiter plate.Thereby, an arrangement of 8×12 wells at a (center-to-center) distanceof about 9 mm is established as the industrial standard. Smaller arrayswith, for example, 3, 6, 12, 24 and 48 wells, arranged at the samedistance, are compatible with this standard. Several arrangements ofsample compartments, according to the invention, provided as smallerarrays of flow cells, can also be combined in such a way, that theindividual inlets of the flow cells are located at a whole-numberedmultiple of the distance of about 9 mm.

Recently, also plates with 384 and 1536 wells, as a whole-numberedmultiple of 96 wells on the same foot print at a correspondingly reducedwell-to-well distance, are used, which shall also be called standardmicrotiter plates. By adaptation of the pitch of the sample compartmentsin the arrangement according to the invention, including the in- andoutlets of each flow cells, to these standards, numerous commerciallyestablished and available laboratory pipettors and robots can be usedfor sample supply.

It is preferred that the exterior dimensions of the arrangementaccording to the invention correspond to the foot print of thesestandard microtiter plates.

Whereas the sample supply is performed in the center of the open samplecompartments in case of commercially available microtiter plates, it isadvantageous for the arrangement according to the invention, due tophysical reasons, if the in- and outlets and the corresponding inlet andoutlet openings are arranged at the border of the corresponding flowcells, for example at corner points opposite to each other, preferably,however, at the diagonally opposite corner points, as it is shown in oneof the examples of break-down-to-praxis. Therefore, the position of thewhole array, of e.g. 8×12 cells in case of an arrangement of 96 flowcells, is preferably slightly displaced from the position of the cellsof a classical microtiter plate with respect to its foot print, thusallowing to address the inlets and/or reservoirs using standardlaboratory robots, without the requirement for re-programming. This isachieved with a displacement of 4.5 mm with respect to the two mainborders (axes) in case of the 96-well pitch, and, correspondingly, of2.25 mm, in case of the 384-well pitch, and of 1.125 mm, in case of the1536-well pitch. Due to technical reasons (available wall thickness ofthe arrangement) a displacement of 2.25 mm is preferred, such that thearrangement according to the invention can be addressed by robotsprovided for the 384-standard, without a modification of theirprogramming.

A further subject of the invention is an arrangement with, for example,2 to 8 sample compartments in a column, with the properties as describedabove, or, for example, 2 to 12 sample compartments in a row, whichthemselves are combined with a carrier (“meta-carrier”) with thedimensions of standard microtiter plates in such a way, that the pitch(geometrical arrangement in rows and/or columns) of the inlets of theflow cells does correspond to the pitch (geometrical arrangement) of thewells of a standard microtiter plate.

The adjoining of the arrangement of sample compartments with themeta-carrier can, for example, be performed by glueing or by exactfitting without glueing, if it is intended for single-use, or, forexample by latching or inserting, if it is intended for multiple use.The material of the meta-carrier can, for example, be selected from thegroup comprising formable, moldable or millable plastics, metals,silicates, such as glass, quartz or ceramics.

Several rows or columns of such sample compartments can also be combinedwith a single meta-carrier in such a way that the pitch (geometricalarrangement in rows and/or columns) of the inlets of the flow cellscorresponds to the pitch (geometrical arrangement) of the wells of astandard microtiter plate, i.e., to a whole-numbered multiple of 9 mm(corresponding to a 96-well plate) or of 4.5 mm (corresponding to a384-well plate, see above) or of 2.25 mm (corresponding to a 1536-wellplate, see above).

Of course, the arrangement of sample compartments according to theinvention can also be arranged in another pitch (geometry). Liquidexiting a flow cell, for example after sequential addition of differentreagents, can be collected in the reservoir fluidically connected withthe corresponding flow cell or also be removed by exhausting at theposition of the inlet or of the reservoir.

There are several technical solutions for the generation of the spatialrecesses between the base plate and the body combined therewith. In onepossible arrangement, three-dimensional structures, with the pitch(geometrical arrangement in rows and/or columns) of the arrays of flowcells to be generated, are formed on the base plate. These structures onthe base plate can, for example, form the walls or parts of the walls,such as sockets, between flow cells adjacent to each other, which flowcells are formed by combination of the base plate with an adequatelyformed body. For generation of the array of flow cells, that, forgeneration of the spatial recesses between the base plate and the bodycombined therewith, these recesses are formed in the base plate.

A characteristic for another embodiment is, that, for generation of therecesses between the base plate and the body combined therewith,recesses are formed in the body. For this embodiment, it is preferredthat the base plate is essentially planar.

The body to be combined with the base plate for the generation of thearray of flow cells can consist of a single workpiece. In anotherembodiment, the body combined with the base plate is formed from severalparts, wherein the combined parts of the body preferably form anirreversibly combined unit.

It is preferred that the body combined with the base plate comprisesauxiliary means facilitating the combination of the body and the baseplate.

It is further preferred that the arrangement comprises a multitude,i.e., 2-2000, preferably 2-400, most preferably 2-100 flow cells.

Preferably, the pitch (geometrical arrangement in rows and/or columns)of the inlets of the flow cells corresponds to the pitch (geometricalarrangement) of the wells of a standard microtiter plate.

A characteristic for another embodiment of the arrangement is that it isclosed by a covering top, such as a foil, membrane or cover plate.

The capacity of the flow cells can be varied within a large range uponvariation of the size of the base areas and of the depth of thetrecesses, so that the inner volume of each flow cell is typically 0.1μl-1000 μl, preferably 1 μl-20 μl. Thereby, the inner volumes ofdifferent flow cells can be similar or different.

If different sample or reagent solutions are filled sequentially into aflow cell, typically a multiple liquid volume of the cell volume isapplied, in order to displace as completely as possible the liquid andcomponents dissolved therein, which had been added before. Therefore, itis preferred that the capacity of the reservoir fluidically connected tothe flow cell is larger, preferably at least 5 times larger than theinner volume of the flow cell.

It is preferred that the depth of the recesses between the base plateand the body combined with the base plate is 1-1000 μm, preferably20-200 μm. The size of the recesses of an array can be uniform ordiverse and the base areas can have any geometry, preferably rectangularor polygonal or also other geometry. The lateral dimensions of the baseareas can be varied within a large range as well, wherein typically thebase areas of the recesses between the base plate and the body combinedwith the base plate are 0.1 mm²-200 mm², preferably 1 mm²-100 mm². It ispreferred, that the corners of the base areas are rounded. Roundedcorners affect the flow profile in a favorable way and facilitate theremoval of gas bubbles that might be formed, and respectively preventtheir formation.

The materials for the baseplate, the body combined therewith, and theoptional additional covering top have to satisfy the requirements of theactually intended application. Dependent on the specific application,these requirements are related to chemical and physical stability, forexample upon exposure to acidic or basic media, salts, alcohols ordetergents as parts of aqueous solutions, or to formamide, to stabilityupon temperature variations (e.g. between −30° C. and 100° C.), tothermal expansion coefficients of the base plate and of the bodycombined therewith as similar as possible, to optical properties (suchas non-fluorescence, reflectivity), to mechanical workability, etc. Itis preferred that the material of the body combined with the base plateis selected from the group formed by formable, moldable or millableplastics, metals, silicates, such as glass, quartz or ceramics. Also thematerial of the additional, contiguous covering top can be selected fromthe group formed by formable, moldable or millable plastics, metals,silicates, such as glass, quartz or ceramics. Concerning the base plate,it is also preferred that its material comprises materials from thegroup formed by formable, moldable or millable plastics, metals,silicates, such as glass, quartz or ceramics. Thereby, theaforementioned components (base plate, body combined therewith, coveringtop) can be composed of a uniform material or can comprise a mixture ora composition adjoined in layers or laterally of different materials,wherein the materials can substitute each other.

A characteristic for a preferred embodiment is that biological orbiochemical or synthetic recognition elements for the determination ofone or more analytes are immobilized on the base plate.

The simplest method of immobilization consists in physical adsorption,for example due to hydrophobic interaction between the recognitionelements and the base plate. However, the extent of these interactionscan be affected strongly by the composition of the medium and itsphysical-chemical properties, such as polarity and ionic strength.Especially in case of sequential addition of different reagents in amulti-step assay, the adhesion of the recognition elements on thesurface, after only adsorptive immobilization, is often insufficient. Inan advanced embodiment of the arrangement, the adhesion is improved bydeposition of an adhesion-promoting layer (f) (according to FIG. 4) onthe base plate for the immobilization of the biological or biochemicalor synthetic recognition elements. It is preferred that theadhesion-promoting layer (f) has a thickness of less than 200 nm,preferably of less than 20 nm. For the generation of theadhesion-promoting layer, many materials can be used. Without anyrestriction, it is preferred that the adhesion-promoting layer (f)comprises chemical compounds from the groups of silanes, epoxides,“self-organized functionalized monolayers”, functionalized polymers andpolymer gels.

In case of the simultaneous determination of different analytes,preferably upon their binding to different, selective recognitionelements, it is of advantage, if the registration of these bindingevents can be performed by detection of laterally resolved signals. Acharacteristic of an advanced embodiment of the arrangement according tothe invention is that the biological or biochemical or syntheticrecognition elements are immobilized in discrete (laterally separated)measurement areas (d). These discrete measurement areas can be formed byspatially selective deposition of the biological or biochemical orsynthetic recognition elements on the base plate. Numerous methods canbe used for the deposition. It is preferred without any restriction ofgenerality that the biological or biochemical or synthetic recognitionelements are deposited on the base plate by one or more methods from thegroup of methods comprising “ink jet spotting, mechanical spotting bymeans of pin, pen or capillary, “micro contact printing”, fluidicallycontacting the measurement areas with the biological or biochemical orsynthetic recognition elements upon their supply in parallel or crossedmicro channels, upon exposure to pressure differences or to electric orelectromagnetic potentials.

As the biological od biochemical or synthetic recognition elements,components from the groups formed by nucleic acids (DNA, RNA) or nucleicacid analogues (for example peptide nucleic acids PNA), antibodies,aptamers, membrane-bound and isolated receptors, their ligands, antigensfor antibodies, cavities generated by chemical synthesis to hostmolecular imprints, and “histidin-tag components”, are deposited. Alsowhole cells and cell fragments can be deposited as biological orbiochemical or synthetic recognition elements.

The arrangement according to the invention is intended for numerousapplications, wherein not only a single, but two or more analytes in asample shall be determined. Therefore, it is preferred that arrays oftwo or more discrete measurement areas (d) each are arranged in theregions of the recesses between the base plate and the body combinedwith the base plate, in which measurement areas similar or differentbiological or biochemical or synthetic recognition elements areimmobilized.

In general, the immobilized recognition elements are selected in such away that they recognize and bind the analyte to be determined with aspecificity as high as possible. Typically however, it must be expectedthat also a nonspecific adsorption of analyte molecules on the surfaceof the base plate occur especially if there are still empty sitesbetween the recognition elements immobilized in the measurement areas.Therefore, it is preferred that compounds which are “chemically neutral”towards the analyte are deposited between the discrete measurement areas(d), in order to reduce nonspecific binding or adsorption. As“chemically neutral” compounds such components are meant, which do notshow a recognition or binding of the analyte, such that no or onlyminimum nonspecific binding occurs. The choice of these compounds isdependent on the properties of the analyte. Without any restriction itis preferred that the “chemically neutral” compounds are selected fromthe groups comprising, for example, albumins, especially bovine andhuman serum albumin, herring sparm or also polyethylen glycols.

The arrangement according to the invention can be used for thedetermination of numerous different parameters (measurands), wherein thespecific embodiment, especially of the baseplate, is dependent on theactually applied measurement method. One subject of the invention is anarrangement, wherein the base plate with the biological or biochemicalor synthetic recognition elements immobilized thereon is operable forthe determination of a change of optical, electrical, electrochemical orthermal parameters, or for the determination of radioactive radiation.It is preferred that the base plate with the biological or biochemicalor synthetic recognition elements immobilized thereon is operable forthe determination of a change of optical parameters, and that the baseplate is transparent in at least one region of wavelengths in thevisible or near-infrared spectral region. It is especially preferredthat the base plate comprises a supporting substrate of glass or of athermoplastic or moldable plastic, which is transparent in at least oneregion of wavelengths in the visible or near-infrared spectral region.

Within the optical detection methods, the methods based on analytedetection within the evanescent field of a waveguide are characterizedby improved sensitivity and by restriction of the detection volume tothe layer penetrated by the ecvanescent field, in proximity to thewaveguide. Therefore, it is preferred that the base plate comprises anoptical waveguide, which is continuous or partitioned into discreteareas. Thereby, especially preferred is an arrangement, wherein theoptical waveguide is an optical film waveguide with a first opticallytransparent layer (a), facing the recesses, on a second opticallytransparent layer (b) with lower refractive index than layer (a)(according to FIG. 4). Thereby, the terminus “optical transparency”means (here and in the following) transparency at one or more excitationwavelengths in the visible or near-infrared spectral range.

The optically transparent layer (b) should be characterized by lowabsorption and fluorescence, in the ideal case free of absorption andfluorescence. Additionally, the surface roughness should be low, becausethe surface roughness of the layer (b) does affect, dependent on thedeposition process to a more or less large extent, the surface roughnessof an additional layer (a) of higher refractive index, when it isdeposited on layer (a) as a waveguiding layer. An increased surfaceroughness at the boundary (interface) layers of layer (a) leads toincreased scattering losses of the guided light, which, however, isundesired. These requirements are fulfilled by numerous materials. It ispreferred that the material of the second optically transparent layer(b) comprises silicates, such as glass or quartz, or a transparentthermoplastic or moldable plastic, preferably of the group comprisingpolycarbonate, polyimide, or polymethylmethacrylate, or polystyrene.

For a given layer thickness of the optically transparent layer (a), thesensitivity of an arrangement according to the invention increases alongwith an increase of the difference between the refractive index of layer(a) and the refractive indices of the adjacent media, i.e., along withan increase of the refractive index of layer (a). It is preferred thatthe refractive index of the first optically transparent layer (a) ishigher than 1.8.

Another important requirement on the properties of layer (a) is that thepropagation losses of the light guided in layer (a) should be as low aspossible. It is preferred that the first optically transparent layer (a)comprises TiO₂, ZnO, Nb₂O₅, Ta₂O₅, HfO₂, or ZrO₂, preferably TiO₂, Ta₂O₅or Nb₂O₅. Combinations of several such materials can also be used.

For a given material of layer (a) and given refractive index, thesensitivity increases with decreasing layer thickness, up to a certainlower limiting value of the layer thickness. The lower limiting value isdetermined by the cut-off of light guiding, if the layer thickness fallsbelow a threshold value determined by the wavelength of the light to beguided, and by an observable increase of the propagation losses in verythin layers, with further decrease of their thickness. It is preferredthat the thickness of the first optically transparent layer (a) isbetween 40 and 300 nm, preferably between 100 and 200 nm.

If an autofluorescence of layer (b) cannot be excluded, especially if itcomprises a plastic such as polycarbonate, or for reducing the effect ofthe surface roughness of layer (b) on the light guiding in layer (a), itcan be advantageous, if an intermediate layer is deposited betweenlayers (a) and (b). Therefore, it is characteristic for anotherembodiment of the arrangement according to the invention, that anadditional optically transparent layer (b′) (according to FIG. IV) withlower refractive index than and in contact with layer (a), and with athickness of 5 nm-10 000 nm, preferably of 10 nm-1000 nm, is locatedbetween the optically transparent layers (a) and (b).

Many methods are known for the in-coupling of excitation light into anoptical waveguide. In case of relatively thick waveguiding layers, up toself-supporting waveguides, the light can be focused into a front end(distal end) of the waveguide, using lenses of adequate numericalaperture, in such a way, that it is guided by internal total reflection.In case of waveguides with larger width of the front face thanthickness, then preferably cylindrical lenses are used. Thereby, thelenses can be arranged remote from the waveguide or can also be directlyconnected to it. This method of front face coupling is less suited incase of lower thicknesses of the waveguiding layer. A more adequatemethod is then the coupling by prisms, which are preferably connected tothe waveguide without any intermediate spacing or mediated by anindex-matching fluid. It is also possible to supply the excitation lightto the optical waveguide of the arrangement according to the inventionby means of an optical fiber, or to couple light that has beenin-coupled into another waveguide into the waveguide of saidarrangement, upon bringing both waveguides in such a proximity to eachother, that their evanescent fields overlap, thus enabling the energytransfer. Therefore, a part of the arrangement according to theinvention is that the in-coupling of excitation light into the opticallytransparent layer (a), to the measurement areas (d), is performed usingone or more optical in-coupling elements from the group comprising prismcouplers, evanescent couplers comprising joined optical waveguides withoverlapping evanescent fields, front face (distal end) couplers withfocusing lenses arranged in front of a front face (distal end) of thewaveguiding layer, and grating couplers.

It is preferred that the in-coupling of excitation light into theoptically transparent layer (a), to the measurement areas (d), isperformed using one or more grating structures (c) (according to FIG.4), that are formed in the optically transparent layer (a).

A further part of the invention is, that the out-coupling of lightguided in the optically transparent layer (a) is performed using gratingstructures (c′) (according to FIG. 4), that are formed in the opticallytransparent layer (a).

Thereby, grating structures (c) and (c′) formed in the opticallytransparent layer (a) can have the same or different periodicity and canbe arranged in parallel or not in parallel to one another.

The arrangement can be provided in such a way that grating structures(c) and (c′) can interchangeably be used as in-coupling and/orout-coupling gratings.

It is preferred that the grating structures (c) and optional additionalgrating structures (c′) have a period of 200 nm-1000 nm and a gratingmodulation depth of 3 nm-100 nm, preferably of 10 nm-30 nm.

Thereby, it is preferred that the ratio of the modulation depth to thethickness of the first optically transparent layer (a) is equal orsmaller than 0.2.

The grating structure can be provided in different forms (geometry). Itis preferred that the grating structure (c) is a relief grating with anyprofile, such as rectangular, triangular or semi-circular profile, or aphase or volume grating with a periodic modulation of the refractiveindex in the essentially planar optically transparent layer (a).

A characteristic for an advanced embodiment of the arrangement is that athin metal layer, preferably of gold or silver, optionally on anadditional dielectric layer of lower refractive index than layer (a),for example of silica or magnesium fluoride, is deposited between theoptically transparent layer (a) and the immobilized biological orbiochemical recognition elements, wherein the thickness of the metallayer and the optional, additional intermediate layer is selected insuch a way that a surface plasmon at the excitation wavelength and/or atthe luminescence wavelength can be excited.

For one embodiment of the arrangement it is preferred that the gratingstructure (c) is a diffractive grating with a uniform period.

For specific applications however, for example for in-couplingsimultaneously excitation light of different wavelengths, it can be ofadvantage if the grating structure (c) is a multi-diffractive grating.

Usually it is preferred that grating structures (c) and optionaladditional grating structures (c′) are located within the region of thesample compartments.

If however, for example, very small sample volumes shall be applied on avery small base area, it can be advantageous if grating structures (c)and optional additional grating structures (c′) are located outside theregion of the sample compartments.

For applications, wherein a number as high as possible, of samplecompartments within one sample compartment, shall be provided, andwherein simultaneously a propagation of the guided excitation light toadjacent sample compartments, in the direction of propagation of theguided excitation light, shall be prevented by means of its controlledoutcoupling, it is preferred that the grating structures (c) are locatedwithin the region of the sample compartments, and that optionaladditional grating structures (c′) are located outside of the samplecompartment, within which the incoupling of the excitation light isperformed.

Especially in case of sequential execution of measurements in differentsample compartments, the gratings structures can be arranged in such away that the excitation light is in-coupled by a grating structure (c)within one sample compartment, propagates through the sample compartment(guided in the waveguiding layer), and enters the waveguiding layerunder an adjacent sample compartment, located in direction ofpropagation of the guided excitation light, where it is out-coupled bymeans of a grating structure (c′) provided therein. After lateraldisplacement of the arrangement, the last-mentioned grating structure(c′) can be used as in-coupling grating itself for a subsequentmeasurement.

Also additional embodiments of the arrangement according to theinvention are provided, wherein grating structures (c) and optionaladditional grating structures (c′) extend over the range of multiple orall sample compartments.

Multiple grating structures (c) and (c′) can also be located within onesample compartment, for sequential measurements within a single samplecompartment.

For most applications of the arrangement, it is preferred that thematerial of the body combined with the baseplate, in the incumbentsurface area on the base plate, is optically transparent, both for theexcitation radiation and for one or more optionally excited luminescenceradiations, at least within the penetration depth of the evanescentfield.

In an advanced embodiment, the material of the body combined with thebase plate is provided in form of a two-layer system, the first layer ofwhich, to be brought into contact with the surface of the base plate,being transparent both for the excitation radiation and optionally forone or more excited luminescence radiations, whereas the adjacent layer,being located more remote from the base plate, is absorbent in thespectral range of the excitation radiation and of the optionally excitedluminescence radiations.

If an optical cross-talk of excitation light between adjacent samplecompartments shall be minimized, it is advantageous to interrupt orminimize (as far as possible) the waveguiding upon contacting thewaveguide in the intermediate regions between the sample compartmentswith an absorbent material. This is especially advantageous, if lightin- and outcoupling by grating structures (c), respectively (c′), isperformed within the sample compartments, or if a large-area gratingstructure extends over a multitude of sample compartments and a largearea is illuminated with excitation light. It is characteristic for suchan advanced embodiment, that the material of the layer in contact withthe base plate is absorbent in the spectral range of the excitationradiation and of the optionally excited luminescence radiations.

It is of advantage if the material of the layer in contact with the baseplate is self-adhesive and tightly sealing. Thereby, it is preferredthat the material of the layer in contact with the base plate comprisesa polysiloxane.

The arrangement according to the invention is operable to determinesimultaneously a multitude of analytes in one sample. Therefore, it isadvantageous if 5-1000, and preferably 10-400 measurement areas arelocated in one sample compartment.

For an advanced embodiment, it is preferred that optically ormechanically recognizable marks are provided on the base plate, in orderto facilitate the adjustment in an optical system and/or to facilitatethe combination of the base plate with the body comprising the recessesfor the sample compartments.

A further subject of the invention is an analytical system for thedetermination of one or more analytes, comprising

-   -   an arrangement according to any of the aforementioned        embodiments    -   means for the locally addressed supply of samples or reagents to        the sample compartments of the arrangement, and        at least one detector for the detection of a change of a        parameter (measurand) due to the presence of the one or more        analytes, which parameter (measurand) preferably is an optical,        electrical, electrochemical or thermal parameter (measurand) or        a signal from radioactive radiation.

A subject of the invention is also an analytical system for thedetermination of one or more luminescences comprising

-   -   an arrangement according to any of the aforementioned        embodiments    -   means for the locally addressed supply of samples or reagents to        the sample compartments of the arrangement    -   at least one excitation light source, and        at least one detector for the detection of the light emanating        from the at least one or more measurement areas (d) on the base        plate.

Preferred is an analytical system for the determination of one or moreanalytes, comprising

-   -   an arrangement according to any of the aforementioned        embodiments    -   means for the locally addressed supply of samples or reagents to        the sample compartments of the arrangement    -   at least one excitation light source, and        at least one detector for the detection of a change of an        optical parameter (measurand), which is preferably a change of        the refractive index and/or of one or more luminescences in the        vicinity of the one or more analytes.

A characteristic of a possible embodiment of the analytical system isthat the launching of the excitation light to the measurement areas (d)is performed in a surface or transmissive illumination configuration.

For specific applications, it is of advantage if the launching of theexcitation light to the measurement areas (d) and the detection of themeasurement light from the measurement areas (d) is performed atopposite sides of the base plate.

For a larger number of applications, it is preferred that the launchingof the excitation light to the measurement areas (d) and the detectionof the measurement light from the measurement areas (d) is performed atthe same side of the base plate.

A characteristic of a special embodiment is that the launching of theexcitation light to the measurement areas (d) and the detection of themeasurement light from the measurement areas (d) is performed in aconfocal arrangement.

For another embodiment of the analytical system according to theinvention, with an arrangement of sample compartments according to theinvention, comprising an optical film waveguide, it is preferred thatthe excitation light rays of one (common) wavelength are located in acommon plane, which is defined by the resonance angle for in-couplingthe excitation light of the excitation wavelength into the opticallytransparent layer (a) by an optical coupling element.

For the simultaneous detection of signals from a multitude ofmeasurement areas, it is preferred that at least one laterally resolvingdetector is used for signal detection, which is preferably selected fromthe group formed by CCD cameras, CCD chips, photodiode arrays, Avalanchediode arrays, multi-channel plates, and multi-channel photomultipliers.

The invention encompasses analytical systems, the characteristics ofwhich is that optical components of the group comprising lenses or lenssystems for the shaping of the transmitted light bundles, planar orcurved mirrors for the deviation and optionally additional shaping ofthe light bundles, prisms for the deviation and optionally spectralseparation of the light bundles, dichroic mirrors for the spectrallyselective deviation of parts of the light bundles, neutral densityfilters for the regulation of the transmitted light intensity, opticalfilters or monochromators for the spectrally selective transmission ofparts of the light bundles, or polarization selective elements for theselection of discrete polarization directions of the excitation orluminescence light are located between the one or more excitation lightsources and the base plate, as part of an arrangement according to anyof the aforementioned embodiments, and/or between the base plate and theone or more detectors.

The light excitation can be performed continuously. It is preferred,however, that the excitation light is launched in pulses with durationof 1 fsec to 10 min.

A characteristic of an advanced embodiment of the analytical system isthat the emission light from the measurement areas is measuredtime-resolved.

It is preferred that for referencing the available excitation light,light signals of the group comprising excitation light at the locationof the light sources or after expansion of the excitation light or afterits multiplexing into individual beams, scattered light at theexcitation wavelength from the location of the one or more discretemeasurement areas, and light of the excitation wavelength outcoupled bythe grating structures (c) or (c′) are measured.

It is especially preferred that the measurement areas for determinationof the emission light and of the reference signal are identical.

In one embodiment of the analytical system according to the invention,launching and detection of the emission light is performedsimultaneously for all measurement areas. A characteristic of anotherembodiment is that launching of the excitation light and detection ofthe emission light from the one or more measurement areas is performedsequentially for one or more sample compartments. It is also possiblethat sequential launching of the excitation light and detection of theemission light from one or more measurement areas is performed severaltimes within a single sample compartment.

Thereby, it is preferred that sequential excitation and detection isperformed using movable optical components of the group comprisingmirrors, deviating prisms, and dichroic mirrors.

It is preferred, that sequential excitation and detection is performedusing an essentially focus and angle preserving scanner.

A characteristic of another embodiment of an analytical system withsequential excitation and detection is that the arrangement, accordingto any of the aforementioned embodiments, is moved between steps ofsequential excitation and detection.

A further subject of the invention is a method for manufacturing anarrangement of sample compartments in a one- or two-dimensional array,comprising a base plate and a body combined with the base plate in sucha way that an array of (spatial) recesses for generation of an array offlow cells, each with at least one inlet and one outlet, are formedbetween the base plate and the body, wherein at least one outlet of eachflow cell is joined with a reservoir fluidically connected with the flowcell, the reservoir being operable to receive liquid exiting the flowcell, wherein the base plate and the body are combined in such a waythat different ones of the (spatial) recesses are fluidically sealedagainst one another.

As a possible embodiment of this method, the base plate and the bodycombined with the base plate can be joined irreversibly. Thereby, it ispreferred that the base plate and the body combined with the base plateare glued together.

Thereby, a glue is preferred which is characterized by transparency ashigh as possible, at least at the excitation wavelength, and isfluorescent as weak as possible under the excitation conditions, in theideal case free of fluorescence. For applications, wherein theexcitation light shall be restricted to a single sample compartment,however, it can also be advantageous if the glue is absorbent at theexcitation wavelength, for example black, but again fluorescent as lowas possible, in the ideal case free of fluorescence. Furtheron, similarmaterial requirements hold for the glue as for the material of the bodycombined with the base plate, i.e. chemical and physical stability, forexample upon exposure to acidic or basic media, salts, alcohols, ordetergents as parts of aqueus solutions, or formamide, and temperaturestability. Of course, the glue has to be adapted simultaneously to thechemical surface properties of the materials to be joined. Chemicalreactions with analytes and/or the immobilized recognition elementsshall also not occur.

If the biological or biochemical or synthetic recognition elements forthe analyte determination are deposited on the base plate before thecombination with the body, the compatibility of the necessary gluecuring method with the stability of the recognition elements also has tobe taken into account. This, in general, excludes those glues, whichrequire for curing the application of very short-wavelength UV-radiation(e.g. shorter than 280 nm), of high temperatures (e.g. above 100° C.),especially when required for longer periods (e.g. longer than 2 hours).Thereby, the requirements are generally stricter, when proteins, such asantibodies, are used as recognition elements, than in combination withnucleic acids as recognition elements.

The base plate and the body combined therewith can also be joinedreversibly, for example by latching, enabled by adequate means providedto that body, such as barbed hooks, or by inserting into providedguideways. An essential criterium for the choice of the method for thejoining of the base plate with the body is, that after itsaccomplishment, adjacent sample compartments are fluidically sealedagainst one another. The fluidic sealing can optionally be supported byusing formable sealing materials. For example, the body can be providedas two- or more-component (layer) system, the layer facing the baseplate being formed by an elastic material, or gaskets (“O-rings”) can beused for sealing. Also the application of a diffusion barrier, in formof a recess in the separation wall between adjacent sample compartments,the separation wall being brought into contact with the base plate, canbe intended for this purpose.

Part of the invention is a method for the determination of one or moreanalytes in one or more liquid samples with an arrangement according toany of the aforementioned embodiments and an analytical system accordingto any of the aforementioned embodiments, wherein sample and optionallyadditional reagent solutions are supplied to the sample compartments,and wherein these liquids can exit into a reservoir fluidicallyconnected with the flow cell, as part of the sample compartments.

A characteristic of an advanced embodiment of the method is thatbiological or biochemical or synthetic recognition elements for thedetermination of one or more analytes are immobilized on the base plateof the arrangement, excitation light is directed to the measurementareas on the base plate, and the light emanating from the measurementareas is detected by at least one detector.

Thereby, a method is preferred wherein the base plate comprises anoptical waveguide, which is continuous or partitioned into discreteareas, excitation light is launched into the waveguide using an opticalcoupling element, and measurement light from the measurement areas,which are in optical interaction with the optical waveguide, is detectedby one or more detectors.

Especially preferred is a method, wherein the optical waveguide isprovided as an optical film waveguide, with a first opticallytransparent layer (a) on a second optically transparent layer (b) withlower refractive index than layer (a), wherein furthermore excitationlight is in-coupled into the optically transparent layer (a), by one ormore grating structures formed in the optically transparent layer (a),and directed, as a guided wave, to the measurement areas locatedthereon, and wherein furthermore the luminescence from molecules capableto luminance, which is generated in the evanescent field of the guidedwave, is detected by one or more detectors, and wherein theconcentration of one or more analytes is determined from the intensityof these luminescence signals.

In case of arrangements with grating structures (c) for the in-couplingof excitation light and additionally provided grating structures (c′)for the out-coupling of light guided in the waveguiding layer, thein-coupling can be optimized by directing the excitation lightout-coupled by a grating structure (c′), directly or after raydeviation, by means of mirrors or prisms, and optionally after focusingby means of an adequate lens, onto a detector, such as a photodiodeconnected to an amplifier. Thereby, it is preferred that the excitationlight is out-coupled along the whole width (dimension normal to thedirection of propagation of the guided excitation light in the plane ofthe waveguiding layer) and is focused onto the light-sensitive area ofthe detector. An optimum in-coupling is achieved when the signal of theout-coupled light generated by this detector reaches its maximum.

It is especially preferred that (1) the isotropically emittedluminescence or (2) luminescence that is in-coupled into the opticallytransparent layer (a) and out-coupled by a grating structure (c) orluminescence comprising both parts (1) and (2) is measuredsimultaneously.

Part of the method according to the invention is that for the generationof the luminescence, a luminescent dye or a luminescent nano-particle isused as a luminescence label, which can be excited and emits at awavelength between 300 nm and 1100 nm.

It is preferred that the luminescence label is bound to the analyte or,in a competitive assay, to an analyte analogue or, in a multi-stepassay, to one of the binding partners of the immobilized biological orbiochemical or synthetic recognition elements or to the biological orbiochemical or synthetic recognition elements.

A characteristic of another embodiment of the method is that a second ormore luminescence labels of similar or different excitation wavelengthas the first luminescence label and similar or different emissionwavelength are used.

Thereby, it is preferred that the second or more luminescence labels canbe excited at the same wavelength as the first luminescence label, butemit at other wavelengths.

It is especially advantageous if the excitation and emission spectra ofthe applied luminescent dyes do not overlap or overlap only partially.

A characteristic of another embodiment of the method is that charge oroptical energy transfer from a first luminescent dye, acting as a donor,to a second luminescent dye, acting as an acceptor, is used for thedetection of the analyte.

A characteristic of still another embodiment of the method is thatbesides determination of one or more luminescences, changes of theeffective refractive index on the measurement areas are determined.

In an advanced embodiment of the method, the one or more determinationsof luminescences and/or determinations of light signals at theexcitation wavelength are performed polarization-selective.

It is preferred that the one or more luminescences are measured at apolarization that is different from the one of the excitation light.

Part of the invention is a method according to any of the aforementionedembodiments for the simultaneous or sequential, quantitative orqualitative determination of one or more analytes of the groupcomprising antibodies or antigens, receptors or ligands, chelators or“histidin-tag components”, oligonucleotides, DNA or RNA strands, DNA orRNA analogues, enzymes, enzyme cofactors or inhibitors, lectins andcarbohydrates.

A characteristic of possible embodiments of the method is that thesamples to be examined are naturally occurring body fluids, such asblood, serum, plasma, lymphe or urine or tissue fluids, or egg yolk.

A characteristic of other embodiments is that the samples to be examinedare optically turbid liquids or surface water or soil or plant extractsor bio- or process broths.

The samples to be examined can also be taken from biological tissue.

A further subject of the invention is the use of a method according toany of the aforementioned embodiments for the quantitative orqualitative determination of chemical, biochemical or biologicalanalytes in screening methods in pharmaceutical research, combinatorialchemistry, clinical and preclinical development, for real-time bindingstudies and the determination of kinetic parameters in affinityscreening and in research, for qualitative and quantitative analytedeterminations, especially for DNA- and RNA analytics and for thedetermination of genomic or proteomic differences in the genome, such assingle nucleotide polymorphisms, for the measurement of protein-DNAinteractions, for the determination of control mechanisms for mRNAexpression and for the protein (bio)synthesis, for the generation oftoxicity studies and the determination of expression profiles,especially for the determination of biological and chemical markercompounds, such as mRNA, proteins, peptides or small-molecular organic(messenger) compounds, and for the determination of antibodies,antigens, pathogens or bacteria in pharmaceutical product developmentand research, human and veterinary diagnostics, agrochemical productdevelopment and research, for symptomatic and pre-symptomatic plantdiagnostics, for patient stratification in pharmaceutical productdevelopment and for the therapeutic drug selection, for thedetermination of pathogens, nocuous agents and germs, especially ofsalmonella, prions and bacteria, in food and environmental analytics.

BRIEF DESCRIPTION OF THE DRAWINGS

The arrangement according to the invention is exemplified in thefollowing figures, without restriction of the subject of the invention.

FIG. 1 shows a cross-sectional partial view, comprising the inlet andoutlet of a single flow cell and parts of the adjacent flow cell.

FIG. 2 shows a cross-sectional partial view corresponding to FIG. 1, foranother embodiment of the arrangement according to the invention.

FIG. 3 depicts shows a cross-sectional partial view corresponding toFIG. 1, for still another embodiment of the arrangement according to theinvention.

FIG. 4 depicts a cross-sectional partial view, restricted to the baseplate, for an embodiment with an optical film waveguide as the baseplate.

FIG. 5 shows an arrangement, wherein column-like arrangements of baseplates and the bodies combined therewith (according to the arrangementof FIG. 2), for generation of, for example, 6 columns with 6 flow cellseach, are themselves then inserted into a common carrier(“meta-carrier”).

EXAMPLES Example 1

It is of advantage if the arrangements of sample compartments accordingto the invention are stackable, and if they comprise means for avoidinga contamination of the base plates 4, upon contact with the environment.

The arrangement according to FIG. 1 comprises a base plate 4 and a body6 combined therewith. The body has a recess 3, which forms a spatial(three-dimensional) recess for generation of a flow cell, with an inlet1 and an outlet 2, after combination of the body with the base plate.The recess 3 can have any geometrical base area; for example, it can berectangular. It is preferred that the corners are rounded (not shown inthe figures). The diameters and cross-sectional areas of inlets 1 andoutlets 2 can be identical or different, and can remain constant orchanging between the inlet opening for the liquid addition and theentrance into the three-dimensional recess 3, respectively between theexit from the spatial (three-dimensional) recess 3 and the entrance intothe reservoir 5 fluidically connected to this flow cell.

For a reduction of sample and reagent volumes required for filling theflow cells, it can be advantageous if the inlet section 1 is broadenedin such a way, that also pipet tips of larger diameter can be insertedinto the inlet, and if the inlet section is closed again at its lowerend, except for a narrow opening for the entrance into the recess 3. Thelater entrance opening can additionally be surrounded by a lowsurrounding wall, within the inlet section 1 (not shown). An additionalsealing of the inlet 1 against adjacent reservoirs 5 can be achieved,for example, if a conically shaped (tapered) pipet tip of a soft,elastic material is pressed against a harder wall material of the body 6(especially concerning the walls of the inlet 1), thus providing asealing function.

For enabling the use of larger pipet tips for the filling step, it canbe further advantageous, if the pitch (geometrical arrangement in rowsand columns) of the reservoirs 5 is displaced with respect to the pitchof the flow cells, including their recesses 3 and the related inletopenings of the inlets 1, in such a way that the entrance openings arealways located at the edge or at a corner of a recess 3 (as shown inFIG. 1), but that the walls (as part of the body 6) towards thereservoir 5 are displaced in direction towards the next flow cell. Thisconfiguration can be helpful for avoiding, within the recesses 3, deadvolumes without flow, and for simultaneously keeping low the requiredliquid volumes.

For facilitating the filling step, the inlet 1 can, for example, beshaped conically (tapered), such that a tip inserted into this inlet isguided towards the entrance opening of the recess 3. The insertion canfurther be facilitated by additional, mechanically structured aids, suchas grooves or ripples in a (conical) entrance opening, towards saidentrance opening in direction of the recess 3 Further, concentrical ringstructures in the wall of the inlet can be provided as aids forcentering.

Further, the addition of components, such as detergents, reducing thesurface tension of the supplied liquids and thus reducing the contactangle with the walls of the flow cells, to the supplied sample and toreagent solutions can facilitate the filling process and avoid theformation of air bubbles during the filling step. For the same purpose,it can be helpful if the walls of the body 6 are themselves modified bychemical or physical surface treatment, such as plasm treatment, in sucha way, that the contact angle is reduced and the wettability thusimproved.

Also shown is the inlet 2 respectively the outlet 1 of the adjacent flowcells (in this cross-sectional view). It is preferred that the inlet andthe outlet of a flow cell are always located at opposite corner pointsof the base area of the recess, in case of an essentially rectangularbase area for example at the endpoints of the diagonal.

In order to allow for a more constant filling and fill velocityrespectively surface wetting of the base plate 4 (for avoiding aparabolic flow profile in the cross-sectional plane normal to FIG. 1),it can be of advantage if the recesses 3 have an increased depth indirection normal to the figure plane, i.e. if the flow cross section isnot rectangular or elliptic, but expanded towards the edges. Thus, amore even flow velocity within the cross-sectional distribution can beachieved, and the formation of dead volumes in the border regions canagain be reduced.

Example 2

FIG. 2 shows another embodiment of the arrangement according to theinvention, wherein the reservoir 5 is provided as a recess in theexterior wall of the body combined with the base plate 4. In thisembodiment, liquid exiting the flow cell can enter the reservoir 5, butcannot flow back into the flow cell, as long as the reservoir is notfilled up to the upper edge of the side wall at the site of the liquidexit.

Example 3

FIG. 3 shows a modified version of an arrangement according to FIG. 1,wherein the reservoir 5 is closed on top. As a consequence, liquidcannot escape, by evaporation, from the reservoir as well. This variantis associated with the additional advantage that such an arrangementaccording to the invention can be fluidically sealed completely,especially safe, upon using an additional covering top, such as a foil,a septum or a cover plate. This is of special importance andadvantageous, if, for example, an escape of biologically or chemicallyhazardous molecules or liquids from the arrangement according to theinvention shall be prevented after the use.

With respect to all described examples, the body 6 can consist of asingle or also of several pieces, which are preferably combined to aunit irreversibly.

Example 4

FIG. 4 exemplifies an arrangement according to the invention, whereinthe base plate is provided as an optical film waveguide with biologicalor biochemical recognition elements immobilized thereon. Here, with “g”are denoted the bordering walls of a flow cell, which is generated uponcombination of the base plate with a body 6, according to the examplesdescribed before. Therefore, “g” corresponds to the denoting label “6”in FIGS. 1-3.

On a layer (b), which is transparent at least in a part of the visibleor near-infrared spectral region, first a thin intermediate layer (b′)and then a layer (a) is deposited, the refractive index of which islarger than the refractive indices of layers (b) and (b′). The layers(a) and (b′) are also optically transparent at least in a part of thevisible and near-infrared spectral range. Grating structures (c) and(c′) are provided in form of relief gratings in layer (b), whichgratings are transferred into the layers located above upon theirdeposition. Then an adhesion-promoting layer (f) is deposited on layer(a), which can improve the adhesion of biological or biochemical orsynthetic recognition elements to be immobilized. In the providedexample, the recognition elements are immobilized in discrete (laterallyseparated) measurement areas (d), which can be arranged, according tothis embodiment, both on and between the grating structures (c) and(c′). In this example, the base plate is finally combined with the body(g) (corresponding to “6” according to the denotation for FIGS. 1-3).

Example 5

FIG. 5 shows an arrangement, wherein column-like arrangements of baseplates and the bodies combined therewith (according to the arrangementof Example 2), for generation of six total columns with six flow cellseach, are themselves then inserted into a common carrier(“meta-carrier”). The column-like base plates 4 and bodies 6 combinedtherewith together form insertion modules 7, which are inserted into themeta-carrier 8.

In the shown example, the meta-carrier has the foot print of amicrotiter plate. The inlet openings 9 towards the inlets 1 (not shownin this figure) are positioned in such a way, that they are compatiblewith the pitch (geometrical arrangements in rows and columns) of a96-well standard microtiter plate, i.e., they are always positioned atintervals of a whole-numbered multiple of 9 mm (in this example:interval of the inlets within a column: 9 mm; interval of the inletsbetween adjacent columns: 18 mm). Upon an adequate displacement of themeta-carrier, together with the insertion modules, the geometricalarrangement of the reservoirs 5 is compatible with the pitch of astandard 96-well microtiter plate correspondingly.

In the shown example, the meta-carrier is provided in such an embodimentthat it can receive up to six insertion modules. However, sites forinsertion modules can also remain vacant.

The insertion of the insertion modules into the meta-carrier, optionallyalso in an automated way, can be facilitated by mechanical aids, such asmechanical limit points or walls, aids for centering, such as mechanicalguides or optical marks.

The adjoining of the arrangement of sample compartments with themeta-carrier can, for example, be performed by glueing or by exactfitting without glueing, if it is intended for single-use, or, forexample by latching or inserting into an adequately formed mounting, ifit is intended for multiple use.

The shown example illustrates an embodiment of a reusable meta-carrier,into which the insertion modules 7 are introduced by means of anattachment mechanism and from where they can be removed again, afterexecution of a measurement.

Preferably, the insertion modules 7 with rows or columns of flow cellsand the corresponding receiving sites of the meta-carrier are providedin such a form that only a single orientation of the insertion modulesis possible upon their insertion.

1. An arrangement of sample compartments in a one-dimensional or atwo-dimensional array, said arrangement comprising: a base plate; and abody combined with said base plate so as to form an array of recessesbetween said base plate and said body, wherein said array of recessesgenerates an array of flow cells fluidically sealed against one another,wherein each of said flow cells includes at least one inlet and at leastone outlet, wherein, for each of said flow cells, said at least oneoutlet is joined with a reservoir fluidically connected with said flowcell, said reservoir being operable to receive liquid exiting from saidat least one outlet of said flow cell, and wherein said reservoir isformed as an enclosed recess in said body.
 2. An arrangement accordingto claim 1, wherein three-dimensional structures with a geometricalarrangement in rows and/or columns of the arrays of flow cells to begenerated, are formed on said base plate.
 3. An arrangement according toclaim 1, wherein, for generation of the array of recesses between saidbase plate and said body combined therewith, recesses are formed in saidbase plate.
 4. An arrangement according to claim 1, wherein, forgeneration of the array of recesses between said base plate and the saidbody combined therewith, recesses are formed in said body.
 5. Anarrangement according to claim 1, wherein said body is made of severalparts which form an irreversibly combined unit.
 6. An arrangementaccording to claim 1, wherein said arrangement comprises 2-2000 flowcells.
 7. An arrangement according to claim 1, wherein a geometricalarrangement in rows and/or columns of the inlets of said flow cellscorresponds to a geometrical arrangement of wells of a standardmicrotiter plate.
 8. An arrangement according to claim 1, with 2 to 8 ofsaid sample compartments in a column or 2 to 12 of said samplecompartments in a row, wherein said sample compartments are combinedwith a meta carrier, said meta carrier having dimensions of a standardmicrotiter plate such that a geometrical arrangement in rows and/orcolumns of said inlets of said flow cells corresponds to a geometricalarrangement of wells of a standard microtiter plate.
 9. An arrangementaccording to claim 1, wherein said arrangement of sample compartments isclosed by covering top.
 10. An arrangement according to claim 1, whereinan inner volume of each flow cell is 0.1 μl-1000 μl.
 11. An arrangementaccording to claim 1, wherein a capacity of said reservoir fluidicallyconnected to said flow cell is at least 5 times larger than an innervolume of said flow cell.
 12. An arrangement according to claim 1,wherein a depth of said recesses between said base plate and said bodycombined with said base plate is 1-1000 μm.
 13. An arrangement accordingto claim 1, wherein base areas of the recesses between said base plateand said body combined with said base plate are 0.1 mm²-200 mm² each,wherein the size of said recesses of an array is uniform or diverse, andwherein the base areas of said recesses are rectangular or polygonal.14. An arrangement according to claim 9, wherein materials of said baseplate, of said body combined with said base plate, and of said coveringtop are selected from formable, moldable or millable plastics, metals,silicates, or ceramics.
 15. An arrangement according to claim 1, whereinbiological or biochemical or synthetic recognition elements for thedetermination of one or more analytes are immobilized on said baseplate.
 16. An arrangement according to claim 1, wherein anadhesion-promoting layer is deposited on said base plate forimmobilization of biological or biochemical or synthetic recognitionelements.
 17. An arrangement according to claim 16, wherein saidadhesion-promoting layer has a thickness of less than 200 nm.
 18. Anarrangement according to claim 16, wherein said adhesion-promoting layercomprises chemical compounds from the groups of silanes, epoxides,self-organized functionalized monolayers, functionalized polymers andpolymer gels.
 19. An arrangement according to claim 1, whereinbiological or biochemical or synthetic recognition elements areimmobilized in discrete measurement areas, and wherein said discretemeasurement areas are formed by spatially selective deposition ofbiological or biochemical or synthetic recognition elements on said baseplate.
 20. An arrangement according to claim 19, wherein the biologicalor biochemical or synthetic recognition elements are deposited on saidbase plate by one or more methods selected from ink jet spotting,mechanical spotting by means of pin, pen or capillary, micro contactprinting, fluidically contacting the measurement areas with thebiological or biochemical or synthetic recognition elements upon theirsupply in parallel or crossed micro channels, upon exposure to pressuredifferences or to electric or electromagnetic potentials.
 21. Anarrangement according to claim 19, wherein, as said biological orbiochemical or synthetic recognition elements, components from thegroups formed by nucleic acids DNA, RNA or nucleic acid analogues,antibodies, aptamers, membrane-bound and isolated receptors, theirligands, antigens for antibodies, cavities generated by chemicalsynthesis to host molecular imprints, and “histidin-tag components”, aredeposited.
 22. An arrangement according to claim 19, wherein, as saidbiological or biochemical or synthetic recognition elements, whole cellsor cell fragments are deposited.
 23. An arrangement according to claim1, wherein arrays of two or more discrete measurement areas each arearranged in regions of said recesses between said base plate and saidbody combined with said base plate, in which measurement areas havingsimilar or different biological or biochemical or synthetic recognitionelements are immobilized.
 24. An arrangement according to claim 19,wherein compounds which are chemically neutral toward an analyte aredeposited between said discrete measurement areas.
 25. An arrangementaccording to claim 24, wherein said chemically neutral compounds areselected from albumins, herring sparm or polyethylen glycols.
 26. Anarrangement according to claim 1, wherein said base plate withbiological or biochemical or synthetic recognition elements immobilizedthereon is operable for the determination of a change of optical,electrical, electrochemical or thermal parameters, or for thedetermination of radioactive radiation.
 27. An arrangement according toclaim 26, wherein said base plate with the biological or biochemical orsynthetic recognition elements immobilized thereon is operable for thedetermination of a change of optical parameters, and wherein said baseplate is transparent in at least one region of wavelengths in thevisible or near-infrared spectral region.
 28. An arrangement accordingto claim 1, wherein said base plate comprises a supporting substrate ofglass or of a thermoplastic or moldable plastic, and wherein said baseplate is transparent in at least one region of wavelengths in thevisible or near-infrared spectral region.
 29. An arrangement accordingto claim 1, wherein said base plate comprises an optical waveguide, saidoptical waveguide being continuous or partitioned into discrete areas.30. An arrangement according to claim 29, wherein said optical waveguideis an optical film waveguide with a first optically transparent layer,facing the recesses, on a second optically transparent layer with lowerrefractive index than said first optically transparent layer.
 31. Anarrangement according to claim 30, wherein a material of the secondoptically transparent layer comprises silicates or a transparentthermoplastic or moldable plastic selected from polycarbonate,polyimide, polymethylmethacrylate, or polystyrene.
 32. An arrangementaccording to claim 30, wherein a refractive index of said firstoptically transparent layer is higher than 1.8.
 33. An arrangementaccording to claim 30, wherein said first optically transparent layercomprises TiO₂, ZnO, Nb₂O₅, Ta₂O₅, HfO₂, or ZrO₁.
 34. An arrangementaccording to claim 30, wherein a thickness of said first opticallytransparent layer is between 40 and 300 nm.
 35. An arrangement accordingto claim 30, wherein an additional optically transparent layer with alower refractive index than said first optically transparent layer andin contact with said first optically transparent layer, and with athickness of 5 nm-10,000 nm, is located between said first and secondoptically transparent layers and.
 36. An arrangement according to claim30, wherein in-coupling of excitation light into said first opticallytransparent layer and to said measurement areas, is performed using oneor more optical in-coupling elements selected from prism couplers,evanescent couplers comprising joined optical waveguides withoverlapping evanescent fields, front face couplers with focusing lensesarranged in front of a front face of a waveguiding layer, and gratingcouplers.
 37. An arrangement according to claim 30, further comprisingone or more grating structures formed in said first opticallytransparent layer, wherein in-coupling of excitation light into saidfirst optically transparent layer and to said measurement areas isperformed using said one or more grating structures.
 38. An arrangementaccording to claim 30, wherein out-coupling of light guided in saidfirst optically transparent layer is performed using said one or moregrating structures that are formed in said first optically transparentlayer.
 39. An arrangement according to claim 37, wherein out-coupling oflight guided in said first optically transparent layer is performedusing said one or more grating structures that are formed in said firstoptically transparent layer, wherein said one or more grating structurescomprises a plurality of grating structures, and wherein said gratingstructures formed in said first optically transparent layer have thesame or different periodicity and are arranged in parallel or not inparallel to one another.
 40. An arrangement according to claim 39,wherein said grating structures can interchangeably be used asin-coupling and/or out-coupling gratings.
 41. An arrangement accordingto claim 37, wherein said one or more grating structures comprises aplurality of grating structure, and wherein said grating structures havea period of 200 nm-1000 nm and a grating modulation depth of 3 nm-100nm.
 42. An arrangement according to claim 41, wherein a ratio of amodulation depth to a thickness of said first optically transparentlayer is equal or smaller than 0.2.
 43. An arrangement according toclaim 37, wherein said one or more grating structures is a reliefgrating, or a phase or volume grating with a periodic modulation ofrefractive index in said first optically transparent layer.
 44. Anarrangement according to claim 30, wherein a thin metal layer disposedon an additional dielectric layer of lower refractive index than saidfirst optically transparent layer is deposited between said firstoptically transparent layer and immobilized biological or biochemicalrecognition elements, and wherein a thickness of said metal layer andsaid additional intermediate layer is selected such that a surfaceplasmon at an excitation wavelength and/or at a luminescence wavelengthcan be excited.
 45. An arrangement according to claim 37, wherein saidone or more grating structures is a diffractive grating with a uniformperiod.
 46. An arrangement according to claim 37, wherein said one ormore grating structures is a multi-diffractive grating.
 47. Anarrangement according to claim 37, wherein said one or more gratingstructures are located within a region of said sample compartments. 48.An arrangement according to claim 37, wherein said one or more gratingstructures are located outside a region of said sample compartments. 49.An arrangement according to claim 37, wherein said one or more gratingstructures extend over a range of multiple or all of said samplecompartments.
 50. An arrangement according to claim 37, wherein said oneor more grating structures comprises a plurality of grating structures,wherein at least one of said grating structures is located within aregion of said sample compartments, and wherein at least one of saidgrating structures is located outside of said sample compartment, withinwhich the in-coupling of excitation light is performed.
 51. Anarrangement according to claim 1, wherein a material of said bodycombined with said base plate, in an incumbant surface area on said baseplate, is optically transparent, both for excitation radiation and forone or more optionally excited luminescence radiations, at least withina penetration depth of an evanescent field.
 52. An arrangement accordingto claim 51, wherein a material of said body combined with the baseplate is provided in a form of a two-layer system, the first layer ofwhich, to be brought into contact with a surface of said base plate,being transparent both for the excitation radiation and for one or moreoptionally excited luminescence radiations, whereas an adjacent layer,being located more remote from said base plate, is absorbent in aspectral range of the excitation radiation and of the optionally excitedluminescence radiations.
 53. An arrangement according to claim 47,wherein a material of a layer in contact with said base plate isabsorbent in a spectral range of an excitation radiation and ofoptionally excited luminescence radiations.
 54. An arrangement accordingto claim 1, wherein a material of a layer of said body in contact withsaid base plate is self-adhesive and tightly sealing.
 55. An arrangementaccording to claim 1, wherein a material of a layer of said body incontact with the base plate comprises a polysiloxane.
 56. An arrangementaccording to claim 1, wherein 5-1000 measurement areas are located inone sample compartment.
 57. An arrangement according to claim 1, whereinoptically or mechanically recognizable marks are provided on said baseplate in order to facilitate adjustment in an optical system and/or tofacilitate the combination of said base plate with said body comprisingsaid recesses for said sample compartments.